The long-term goal of this research project is to advance our understanding of the molecular mechanisms and principles by which the bidirectional signaling interaction between the skeletal muscle dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) controls dynamic changes in myoplasmic Ca2+ during excitation-contraction (EC) coupling in normal and diseased muscle. This project will first test the hypothesis that: Mutations in the DHPR and RyR1 proteins that cause MH and CCD result in "leaky" or "EC uncoupled" SR Ca2+ release channels. Aim #1 will determine the functional properties and structural determinants of leaky and EC uncoupled release channels caused by newly identified MH and CCD mutations in RyR1. Experiments will evaluate the impact of these mutations on resting Ca2+, SR Ca2+content, and electrically-evoked Ca2+ release following expression in dyspedic myotubes. Whole-cell patch clamp experiments will be used to obtain a more detailed characterization of DHPR/RyR1 function. Aim #2 will compare the functional impact of MH and CCD mutations engineered into rabbit and human RyR1 proteins. Experiments are also planned to determine the potential for dantrolene in restoring normal Ca2+homeostasis in dyspedic myotubes expressing leaky human SR Ca2+release channels. Aim #3 will compare the sensitivity of dyspedic myotubes expressing wild-type and CCD mutant channels to activation by mechanistically distinct triggering paradigms (voltage, caffeine, 4-chloro-m-cresol, Ca2+) to test the hypothesis that leaky channel behavior arises from a global destabilization of the resting closed state of the release channel. Aim #4 will evaluate the effects on EC coupling of MH mutations located in the DHPR III-IV loop following expression in dysgenic myotubes. The second phase of this project will test the hypothesis that: ApoCaM and CaCaM modulate skeletal muscle EC coupling by interacting with overlapping binding sites (between residues 3614- 3643) in RyR1. Aim #5 will overexpress Ca2+binding-deficient (CaM1-4, CaM1,2, and CAM3,4 ) and high affinity Ca2+ binding (CaMCC) CaMs in myotubes to assess the impact of CaM on DHPR/RyR1 function and EC coupling. Aim #6 will dissect the role of apoCaM and CaCaM on release channel function and EC coupling by expressing point mutations in RyR1 that disrupt both apoCaM and CaCaM binding (L3624D) or only CaCaM binding (W3620A). This project will employ a multi-disciplinary approach that combines the tools of molecular biology, cell biology, electrophysiology, biochemistry, and fluorescence microscopy to probe the mechanisms of Ca2+ homeostasis and EC coupling in normal and diseased muscle.